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Abstract Polyphenol oxidases (PPOs) present in mature wheat kernerals have been implicated in the undesirable darkening of dough. To accelerate the functional characterization of wheat PPOs, allow the identification of those PPO genes that are primarily involved in food biochemistry, and improve the appearance quality of cereal products of china, basic local alignment search tool (BLAST) searches of expressed sequence tag (EST) databases were performed, and the sequences were aligned using software DNAMAN. Results from this study suggest that the presence of at least 13 PPO sequences in hexapolid wheat fell into two clusters (I、II). Genes in ‘II’ cluster are expressed during kernel development and may therefore influence cereal product quality, and the genes in ‘i’ class of II cluster which can be used in the further molecular marker related research seemed not on the wheat chromosome 2A and 2D. The allelic variations of PPO genes on chromosome 2D (PPO-2Da、b) were richer than PPO genes on chromosome 2A (PPO-2Aa、b) according the sequence alignment between four PPO genes with open reading frame (ORF). 94 single nucleotide polymorphisms (SNP) were detected between PPO-2Da and PPO-2Db and 80 SNPs were found in coding region (coding SNP) while 36 SNPs,which affect the PPO amino acid sequece were non-synonymous cSNPs. To understand the effect of non-synonymous cSNPs in PPO gene on grain PPO activity, primer was designed as STS-H at some non-synonymous cSNPs site. The STS-H can amplify a 460-bp fragment in most cultivars with high PPO activity (b), while no PCR product was detected in most cultivars with low PPO activity (a). A total of 130 wheat cultivars were used to validate the correlation between the polymorphic fragments of STS-H and grain PPO activity. The results showed that PPO alleles ‘a’ and ‘b’ at the STS-H locus gave significantly different effect on grain PPO activity. The cultivars with allele ‘b’ had greater (P<0.01) PPO activity than the cultivars with allele ‘a’. To enhance the selection efficiency of single dominance molecular marker, the multiplex PCR was developed between STS-H and STS01, which is a STS molecular marker for low PPO activity and complementary with STS-H tested in this study.
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Received: 10 April 2007
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